Structures of the holoenzyme TglHI required for 3-thiaglutamate biosynthesis

Structural diverse natural products like ribosomally synthesized and posttranslationally modified peptides (RiPPs) display a wide range of biological activities. Currently, the mechanism of an uncommon reaction step during the biosynthesis of 3-thiaglutamate (3-thiaGlu) is poorly understood. The removal of the β-carbon from the Cys in the TglA-Cys peptide catalyzed by the TglHI holoenzyme remains elusive. Here, we present three crystal structures of TglHI complexes with and without bound iron, which reveal that the catalytic pocket is formed by the interaction of TglH–TglI, and that its activation is conformation dependent. Biochemical assays suggest a minimum of two iron ions in the active cluster and we identify the position of a third iron site. Collectively, our study offers insights into the activation and catalysis mechanisms of the nonheme dioxygen-dependent holoenzyme TglHI. Additionally, it highlights the evolutionary and structural conservation in the DUF692 family of biosynthetic enzymes that produce diverse RiPPs.

(B) Role of TglHI in the biosynthesis of 3-thiaGlu.First, TglB forms TglA-Cys by cysteinylating the ribosomally translated precursor peptide TglA (orange) at its phosphorylated C-terminus in an ATP-and Cys-tRNA Cys -dependent manner.After that, the oxygen-dependent excision of the Cys methylene group is catalyzed by the TglHI complex, releasing the Cβ as formate and generating an intermediate containing an alpha-thiol group.The C-terminal secondary thiol is then modified by a carboxy-SAM (Cx-SAM)dependent methyltransferase called TglF to yield TglA-thiaGlu.The C-terminal 3-thiaglutamate is then released by the membrane-associated protease TglG, releasing TglA as a scaffold for the following biosynthesis cycle.tglH and tglI genes were inserted into the pET28b vector and pET21b vector, respectively.tglH-pET28b construct contains both N-terminal and Cterminal His 6 -tags, whereas the tglI-pET21b construct contains only the C-terminal His 6 -tag.When TglH and TglI were co-expressed, the His 6 tag at the C-terminal end of TglI was removed.
(B) The tglH and tglI genes were inserted into the pRSF-DuET-1 vector for co-expression, and the complexes were purified through nickel affinity columns.The full-length TglA-Cys protein carrying the GST tag was purified and was bound to a GST affinity resin.Subsequently, TglA-Cys-bound resin was incubated with the purified TglHI protein for 2 h and then the column was washed.The proteins were then eluted and analyzed by SDS-PAGE.(C) TglH active site variants co-expressed with His 6 -TglA-Cys and TglI followed by Ni-NTA purification and analysis of the peptide by MALDI-TOF MS.The N187A, H216A, and D229A products appear to contain another ion at slightly lower m/z as judged by the wider isotope envelope.(D-E) Attempts to identify the N187A, H216A, and D229A products by S-alkylation with iodoacetamide followed by digestion with trypsin and analysis by HR ESI MS (D) showed only the regular product as demonstrated by tandem MS (E).The identity of the additional mass observed in the MALDI-TOF mass spectrum is therefore unknown and could be derived from a peptide or protein from E. coli.Nevertheless, the data clearly show that these three TglH variants still produce the same product as wild type TglH.(C) The predicted mechanism of engagement of TglA-Cys with TglI.Four residues of TglA-Cys (Phe33, Glu34, Glu35, and Phe36) and TglI form a reverse β-strand through hydrogen bonds (shown in red dashes) between amide N-H and carbonyl oxygens on the backbone.(D) In vitro activity of TglHI mutants involved in the interaction between the minimum substrate analog of TglA-Cys and TglI.(E) In vitro activity of TglHI incubated with the minimum substrate analog of TglA-Cys, TglA-Ser, TglA-Thr and TglA-Val, respectively.The latter three peptides give responses that are indistinguishable from a negative control in which TglHI was omitted (pink) due to formate contamination, which is ubiquitous.(F) Proposed mechanism of TglHI.The proposed carbon excision reaction catalyzed by TglHI begins with the activation of oxygen by Fe 1 that is also liganded by the C-terminal cysteine of TglA-Cys, but we cannot rule out that the oxygen interacts with one of the other irons, which would need to be in the ferrous form.Asn73 is required for activity and is likely important to orient Cys for coordination to Fe 1 by the formation of a hydrogen bond between the -NH 2 of its side chain amide and the thiolate of Cys.This orientation may also be important for hydrogen atom abstraction from the β-carbon of the Cys.As shown the mechanism implies that TglHI catalyzes dioxygenase activity but it has not been experimentally shown that both oxygens in formate are derived from O 2 .The iron-bound hydroxide that hydrolyzes the formyl thioester in the final step could exchange with solvent leading to only one oxygen of O 2 ending up in formate.
Figure S1.3-Thiaglutamate (3-thiaGlu) biosynthesis in Pseudomonas syringae pv.maculicola str.ES4326 ， and sequence alignment of TglH and MbnB required for methanobactin (Mbn) biosynthesis (Related to Figure 1) (A) The tglH (pink) and tglI (slate) genes are colored in the tgl biosynthetic gene cluster; sites encoding RiPP recognition element (RRE) domains are indicated in gray.The minimum substrate analog of TglA is highlighted in orange.(B)Role of TglHI in the biosynthesis of 3-thiaGlu.First, TglB forms TglA-Cys by cysteinylating the ribosomally translated precursor peptide TglA (orange) at its phosphorylated C-terminus in an ATP-and Cys-tRNA Cys -dependent manner.After that, the oxygen-dependent excision of the Cys methylene group is catalyzed by the TglHI complex, releasing the Cβ as formate and generating an intermediate containing an alpha-thiol group.The C-terminal secondary thiol is then modified by a carboxy-SAM (Cx-SAM)dependent methyltransferase called TglF to yield TglA-thiaGlu.The C-terminal 3-thiaglutamate is then released by the membrane-associated protease TglG, releasing TglA as a scaffold for the following biosynthesis cycle.(C) Biosynthesis of Mbn from MbnA catalyzed by MbnBC.The oxazolone-thioamide installation is executed by MbnBC-catalyzed MbnA modification, in which the MbnA Cys sulfhydryl group is ligated to one of the irons of the tri-iron center of dioxygen-dependent MbnBC holoenzyme.(D) Amino acid sequence alignment of TglH, HsMbnB (Histophilus somni, WP_249962398.1),VcMbnB (Vibrio caribbenthicus, WP_009601368.1),RrMbnB (Rugamonas rubra, WP_093383377.1)and MtMbnB (Methylosinus trichosporium, WP_065083569.1).Residues in TglH that involve coordination to Fe are indicated by green triangles (Structural superposition demonstrates that E258 in TglH corresponds to E238 in VcMbnB or E240 in RrMbnB with a black triangle), and a key residue for catalysis is indicated by a green diamond.The Asp that has been proposed to serve as a general base in MbnB is indicated in a black diamond.Fully conserved residues are highlighted in red and mostly conserved residues are highlighted in yellow.For many of the yellow highlighted residues, the amino acid in TglH is different from that in MbnB.
Figure S2.Pull-down assay for the TglHI and TglA-Cys complexes, Interaction between apoTglH and TglI (Related to Figure2) (A) SDS-PAGE of TglH and TglI when expressed separately.tglH and tglI genes were inserted into the pET28b vector and pET21b vector, respectively.tglH-pET28b construct contains both N-terminal and Cterminal His 6 -tags, whereas the tglI-pET21b construct contains only the C-terminal His 6 -tag.When TglH and TglI were co-expressed, the His 6 tag at the C-terminal end of TglI was removed.(B) The tglH and tglI genes were inserted into the pRSF-DuET-1 vector for co-expression, and the complexes were purified through nickel affinity columns.The full-length TglA-Cys protein carrying the GST tag was purified and was bound to a GST affinity resin.Subsequently, TglA-Cys-bound resin was incubated with the purified TglHI protein for 2 h and then the column was washed.The proteins were then eluted and analyzed by SDS-PAGE.(C) In vitro activity assay of the modification of the minimum substrate analog of TglA-Cys by TglHI expressed in E. coli BL21 grown in LB medium supplemented without (green) and with (purple, blue) different concentration of ammonium ferrous sulfate in the presence of formate dehydrogenase (FDH) and β-NAD + .(D) Hydrophobic interface I formed by L1 (residues 41-53) of apoTglH with TglI.The electrostatic potential in all figures was computed using the APBS tools in PyMol (http://www.pymol.org/).(E) Hydrophobic interface II formed by L3 (residues 151-166) of apoTglH with TglI.(F) Interaction between VcMbnB and the N-terminal domain (NTD) of VcMbnC (PDB accession No. 7DZ9).The NTD of MbnC is sandwiched between two extended loops of MbnB.(G) Interaction between VcMbnB and the C-terminal domain (CTD) of VcMbnC (PDB accession No. 7DZ9).The hairpin loop between β2 and β3 of VcMbnC CTD contacts VcMbnB.(H) Coomassie stained SDS-PAGE gel showing co-expressed TglHI, TglH ΔL1 I, TglH ΔL3 I, TglHI △α1 and TglHI △α3 complexes.The TglHI positions are indicated with red boxes.L3 in TglH engages in interactions with α1 and α3 in TglI.(I) Coomassie stained SDS-PAGE gel showing co-expressed TglHI △199-216 and TglHI △CTD , and pull-down assay of these mutant and TglA-Cys.The positions for TglHI mutants are indicated with red boxes.The destruction of the C-terminal domain (residues 152-269, CTD) of TglI in TglHI leads to the disappearance of the TglA-Cys interaction with TglHI.Abbreviations: C, crude; P, pellet; F, flow through; E, eluent; R1, resin before TglHI was added; R2, resin after TglHI addition and washing.

Figure S4 .
Figure S2.Pull-down assay for the TglHI and TglA-Cys complexes, Interaction between apoTglH and TglI (Related to Figure2) (A) SDS-PAGE of TglH and TglI when expressed separately.tglH and tglI genes were inserted into the pET28b vector and pET21b vector, respectively.tglH-pET28b construct contains both N-terminal and Cterminal His 6 -tags, whereas the tglI-pET21b construct contains only the C-terminal His 6 -tag.When TglH and TglI were co-expressed, the His 6 tag at the C-terminal end of TglI was removed.(B) The tglH and tglI genes were inserted into the pRSF-DuET-1 vector for co-expression, and the complexes were purified through nickel affinity columns.The full-length TglA-Cys protein carrying the GST tag was purified and was bound to a GST affinity resin.Subsequently, TglA-Cys-bound resin was incubated with the purified TglHI protein for 2 h and then the column was washed.The proteins were then eluted and analyzed by SDS-PAGE.(C) In vitro activity assay of the modification of the minimum substrate analog of TglA-Cys by TglHI expressed in E. coli BL21 grown in LB medium supplemented without (green) and with (purple, blue) different concentration of ammonium ferrous sulfate in the presence of formate dehydrogenase (FDH) and β-NAD + .(D) Hydrophobic interface I formed by L1 (residues 41-53) of apoTglH with TglI.The electrostatic potential in all figures was computed using the APBS tools in PyMol (http://www.pymol.org/).(E) Hydrophobic interface II formed by L3 (residues 151-166) of apoTglH with TglI.(F) Interaction between VcMbnB and the N-terminal domain (NTD) of VcMbnC (PDB accession No. 7DZ9).The NTD of MbnC is sandwiched between two extended loops of MbnB.(G) Interaction between VcMbnB and the C-terminal domain (CTD) of VcMbnC (PDB accession No. 7DZ9).The hairpin loop between β2 and β3 of VcMbnC CTD contacts VcMbnB.(H) Coomassie stained SDS-PAGE gel showing co-expressed TglHI, TglH ΔL1 I, TglH ΔL3 I, TglHI △α1 and TglHI △α3 complexes.The TglHI positions are indicated with red boxes.L3 in TglH engages in interactions with α1 and α3 in TglI.(I) Coomassie stained SDS-PAGE gel showing co-expressed TglHI △199-216 and TglHI △CTD , and pull-down assay of these mutant and TglA-Cys.The positions for TglHI mutants are indicated with red boxes.The destruction of the C-terminal domain (residues 152-269, CTD) of TglI in TglHI leads to the disappearance of the TglA-Cys interaction with TglHI.Abbreviations: C, crude; P, pellet; F, flow through; E, eluent; R1, resin before TglHI was added; R2, resin after TglHI addition and washing.
Figure S6.Specific recognition of TglA-Cys by TglHI (Related to Figure 5) (A) Structure of TglHIA-Cys complex predicted by AlphaFold2 (named TglHIA-Cys-AF2).(B) Superposition of TglH (top) and TglI (bottom) structures from apoTglHI, TglHI-2Fe and TglHI-AF2, respectively.(C)The predicted mechanism of engagement of TglA-Cys with TglI.Four residues of TglA-Cys (Phe33, Glu34, Glu35, and Phe36) and TglI form a reverse β-strand through hydrogen bonds (shown in red dashes) between amide N-H and carbonyl oxygens on the backbone.(D) In vitro activity of TglHI mutants involved in the interaction between the minimum substrate analog of TglA-Cys and TglI.(E) In vitro activity of TglHI incubated with the minimum substrate analog of TglA-Cys, TglA-Ser, TglA-Thr and TglA-Val, respectively.The latter three peptides give responses that are indistinguishable from a negative control in which TglHI was omitted (pink) due to formate contamination, which is ubiquitous.(F) Proposed mechanism of TglHI.The proposed carbon excision reaction catalyzed by TglHI begins with the activation of oxygen by Fe 1 that is also liganded by the C-terminal cysteine of TglA-Cys, but we cannot rule out that the oxygen interacts with one of the other irons, which would need to be in the ferrous form.Asn73 is required for activity and is likely important to orient Cys for coordination to Fe 1 by the formation of a hydrogen bond between the -NH 2 of its side chain amide and the thiolate of Cys.This orientation may also be important for hydrogen atom abstraction from the β-carbon of the Cys.As shown the mechanism implies that TglHI catalyzes dioxygenase activity but it has not been experimentally shown that both oxygens in formate are derived from O 2 .The iron-bound hydroxide that hydrolyzes the formyl thioester in the final step could exchange with solvent leading to only one oxygen of O 2 ending up in formate.

Figure S7 .
Figure S7.Structures of TIM-barrel-related enzymes (Related to Figure 6)The active cavities of these enzymes are covered by flexible loops, a structural feature similar to that of TglH.This closed cavity, which is essential for enzyme activity, may reflect the evolutionary conservation of the structure.
(C) Biosynthesis of Mbn from MbnA catalyzed by MbnBC.The oxazolone-thioamide installation is executed by MbnBC-catalyzed MbnA modification, in which the MbnA Cys sulfhydryl group is ligated to one of the irons of the tri-iron center of dioxygen-dependent MbnBC holoenzyme.(D) Amino acid sequence alignment of TglH, HsMbnB (Histophilus somni, WP_249962398.1),VcMbnB (Vibrio caribbenthicus, WP_009601368.1),RrMbnB (Rugamonas rubra, WP_093383377.1)and MtMbnB (Methylosinus trichosporium, WP_065083569.1).Residues in TglH that involve coordination to Fe are indicated by green triangles (Structural superposition demonstrates that E258 in TglH corresponds to E238 in VcMbnB or E240 in RrMbnB with a black triangle), and a key residue for catalysis is indicated by a green diamond.The Asp that has been proposed to serve as a general base in MbnB is indicated in a black diamond.Fully conserved residues are highlighted in red and mostly conserved residues are highlighted in yellow.For many of the yellow highlighted residues, the amino acid in TglH is different from that in MbnB.

Table S1 . Fe content analysis for TglHI and TglHIA-Cys (Related to Figures 1 and 4)
The apoTglHI was expressed in in E. coli BL21 grown in M9 medium supplemented without ammonium ferrous sulfate.Other proteins were obtained in LB medium supplemented without and with different concentrations of ammonium ferrous sulfate (1mM, 2mM), respectively.All the values are mean and standard deviation of the measured metal concentrations for three independent protein preparations. a

Table S3 . The recipe of minimal nutrient M9 medium (Related to STAR Methods) Reagent Content (Final concentration in 1L)
YNB medium was from BD Difco.And the other regents were purchased from MilliporeSigma (Darmstadt Germany)